NLSdb: database of nuclear localization signals

NLSdb is a database of nuclear localization signals (NLSs) and of nuclear proteins. NLSs are short stretches of residues mediating transport of nuclear proteins into the nucleus. The database contains 114 experimentally determined NLSs that were obtained through an extensive literature search. Using 'in silico mutagenesis' this set was extended to 308 experimental and potential NLSs. This final set matched over 43% of all known nuclear proteins and matches no currently known non-nuclear protein. NLSdb contains over 6000 predicted nuclear proteins and their targeting signals from the PDB and SWISS-PROT/TrEMBL databases. The database also contains over 12 500 predicted nuclear proteins from six entirely sequenced eukaryotic proteomes (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae). NLS motifs often co-localize with DNA-binding regions. This observation was used to also annotate over 1500 DNA-binding proteins. NLSdb can be accessed via the web site: http://cubic.bioc.columbia.edu/db/NLSdb/.     

Identification and characterization of nuclear localization signals of CaMKP-N

Calmodulin-dependent protein kinase phosphatase (CaMKP) and CaMKP-N dephosphorylate and regulate multifunctional Ca(2+)/calmodulin-dependent protein kinases. The enzymatic properties of CaMKP-N and CaMKP resemble each other, whereas their localizations are different. CaMKP-N is localized in the nucleus, whereas CaMKP is localized in the cytosol. In the present study, the nuclear localization signals (NLSs) of CaMKP-N were identified and characterized. CaMKP-N contains two NLSs, NLS1 and NLS2, at the C-terminus. A cluster of basic residues in the NLSs is important for their function. NLS1 and NLS2 function independently, but mutagenesis analysis suggests that these NLSs interact with each other.     

Cellular localization, half-life, and secretion of peptide YY

Tissue and plasma concentration of peptide YY (PYY) were measured by means of a radioimmunoassay (RIA) developed in our laboratory, using a specific PYY antiserum generated in New Zealand white rabbits against synthetic PYY, and dextran-coated charcoal to terminate the assay. Cellular localization of PYY was studied immunohistochemically using the peroxidase-antiperoxidase (PAP) technique. The highest tissue concentration of PYY was found in the mucosa of the terminal ileum and colon. PYY-containing secretory granules were primarily found in the basal pole of open-type endocrine cells. Basal plasma concentration of PYY was 70 +/- 9 pg/ml and rose to 357 +/- 30 pg/ml during the IV administration of PYY at 400 pmol/kg-h. A significant correlation was found (r = 0.94, p less than 0.05) between dose of PYY (12.5, 25, 50, 100, 200, 400 pmol/kg-h, IV) and plasma concentration of PYY. The calculated half-life of PYY in plasma was 8.3 +/- 1.9 minutes. Plasma concentration of PYY during the intraduodenal administration of sodium oleate (150 +/- 20 pg/ml) or long-chain triglyceride (187 +/- 37 pg/ml) was similar to plasma concentration of PYY obtained during the IV administration of PYY at 100 pmol/kg-h. Plasma concentration of PYY raised (126 +/- 10 pg/ml) after the administration of bombesin (400 pmol/kg-h, IV). Bile enhanced release of PYY. The present study suggests a hormonal role for PYY.     

Modular organization and combinatorial energetics of proline-tyrosine nuclear localization signals

Proline-tyrosine nuclear localization signals (PY-NLSs) are recognized and transported into the nucleus by human Karyopherin (Kap) beta2/Transportin and yeast Kap104p. Multipartite PY-NLSs are highly diverse in sequence and structure, share a common C-terminal R/H/KX2-5PY motif, and can be subdivided into hydrophobic and basic subclasses based on loose N-terminal sequence motifs. PY-NLS variability is consistent with weak consensus motifs, but such diversity potentially renders comprehensive genome-scale searches intractable. Here, we use yeast Kap104p as a model system to understand the energetic organization of this NLS. First, we show that Kap104p substrates contain PY-NLSs, demonstrating their generality across eukaryotes. Previously reported Kapbeta2-NLS structures explain Kap104p specificity for the basic PY-NLS. More importantly, thermodynamic analyses revealed physical properties that govern PY-NLS binding affinity: (1) PY-NLSs contain three energetically significant linear epitopes, (2) each epitope accommodates substantial sequence diversity, within defined limits, (3) the epitopes are energetically quasi-independent, and (4) a given linear epitope can contribute differently to total binding energy in different PY-NLSs, amplifying signal diversity through combinatorial mixing of energetically weak and strong motifs. The modular organization of the PY-NLS coupled with its combinatorial energetics lays a path to decode this diverse and evolvable signal for future comprehensive genome-scale identification of nuclear import substrates.     

Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals

The interaction between phosphatidylserine and its receptor on phagocytic cells plays a critical role in the clearance of apoptotic bodies under normal physiological condition. A specific receptor for phosphatidylserine (PSR) has recently been identified by phage display and shown to mediate phosphatidylserine dependent phagocytosis. Here we show that the protein encoded by the PSR cDNA is localized in the nuclei through multiple nuclear localization signals. First, a fusion between PSR and GFP is localized in the nuclei of transfected cells, suggesting that PSR have intrinsic nuclear localization capability. Indeed, affinity-purified anti-PSR antibodies identified a 47 kDa protein species in cells transfected with untagged PSR and localized this protein in the nuclei by immunofluorescent confocal microscopy. In NIH3T3 cells, which express endogenous PSR mRNA, a similar 47 kDa species was detected and localized in the nuclei. Finally, multiple nuclear localization signals were identified in PSR sequence, each capable of targeting GFP to the nuclei. Together, these results suggest that PSR may serve a dual role both on the cell surface and in the nuclei.     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

细胞质内信号分子的核转位及其机制

细胞外信号通过受体及细胞内信号转导引起细胞生长,增殖,分化,凋亡等细胞核反应。进入细胞质内的信号分子及其活化产物必须经过细胞核膜上的核孔复合体（ＮＰＣ）,在核定位信号的介导下,由特异性的载体转运入核,该过程涉及小分子的ＧＴＰａｓｅ Ｒａｎ蛋白及多种可溶性因子。本文简要综述细胞质内信号分子通过核膜向细胞核内转运的过程及其调控机制。     

A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.     

Characterization of the nuclear localization and nuclear export signals of bovine herpesvirus 1 VP22

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130PRPR133, was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130PRPR133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204LDRMLKSAAIRIL216, was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.     

Functional characterization of nuclear localization signals in yeast Sm proteins

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.     

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.     

Classical nuclear localization signals: definition, function, and interaction with importin alpha

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.